1. Field of the Invention
The present invention relates generally to electrophoresis, and more particularly to a microchip that performs electrophoresis, a method to produce a microchip with integrated electrodes, and a method for performing electrophoresis using a microchip with integrated electrodes.
2. Related Art
Microanalytical devices open up new possibilities for the miniaturization of conventional chemical and biochemical analysis systems. Since the introduction of the Miniaturized Total Analysis System (μTAS) less than 15 years ago, an enormous number of papers have been published showing the capabilities of these devices, see Terry, S. C., Jerman, J. H., Angell, J. B., IEEE Trans. Electron. Devices 1979, ED-26, 1880; Verpoorte, E. Electrophoresis 2002, 23, 677-712; Reyes, D. R., Iossifidis, D., Auroux, P. A., Manz, A., Anal. Chem. 2002, 74, 2623-2636; Auroux, P. A., Iossifidis, D., Reyes, D. R., Manz, A., Anal. Chem. 2002, 74, 2637-2652, the entire contents and disclosures of which are hereby incorporated by reference. Some of the advantages of the μTAS over conventional bench-top systems include custom design, reduced consumption of reagents and sample, lower waste generation and increased analysis speed and portability, see Ng, J. M., Gitlin, I., Stroock, A. D., Whitesides, G. M., Electrophoresis 2002, 23, 3461-3473, the entire contents and disclosure of which is hereby incorporated by reference. Born as the combination of microelectronics technology and capillary electrophoresis, microanalytical devices were initially constructed using silicon or glass substrates. A wide variety of polymeric materials have been used more recently, see Becker, H., Locascio, L., Talanta 2002, 56, 267-287, the entire contents and disclosure of which is hereby incorporated by reference. In particular, poly(dimethylsiloxane) (PDMS) has been used extensively because it is robust, optically transparent, non-polar, impermeable to aqueous solutions and allows the easy, fast, and inexpensive fabrication of devices by using micromolding techniques, see Duffy, D. C., McDonald, J. C., Schueller, O. J. A., Whitesides, G. M., Anal. Chem. 1998, 70, 4974-4984, the entire contents and disclosure of which is hereby incorporated by reference. Although there are some examples of pressure-driven applications, PDMS has been used extensively for microchip electrophoresis with a variety of applications including immunoanalysis, DNA analysis, and small molecule determinations, see Reyes, D. R., Iossifidis, D., Auroux, P. A., Manz, A., Anal. Chem. 2002, 74, 2623-2636; Auroux, P. A., Iossifidis, D., Reyes, D. R., Manz, A., Anal. Chem. 2002, 74, 2637-2652; Garcia, C. D., Hadley, D. G., W. W., W., Henry, C. S., Biotechnol. Prog. 2003, in press; Guijt, R. M., Baltussen, E., van Dedem, G. W., Electrophoresis 2002, 23, 823-835; Linder, V., Verpoorte, E., de Rooij, N. F., Sigrist, H., Thormann, W., Electrophoresis 2002, 23, 740-749; Weigl, B. H., Bardell, R. L., Cabrera, C. R., Adv. Drug. Deliv. Rev. 2003, 55, 349-377; Zhang, L., Dang, F., Baba, Y., J. Pharm. Biomed. Anal. 2003, 30, 1645-1654; Chiari, M., Cretich, M., Consonni, R., Electrophoresis 2002, 23, 536-541; Dolnik, V., Liu, S., Jovanovich, S., Electrophoresis 2000, 21, 41-54; Manica, D. P., Ewing, A. G., Electrophoresis 2002, 23, 3735-3743; Wang, J., Talanta 2002, 56, 223-231; and Rossier, J., Reymond, F., Michel, P. E., Electrophoresis 2002, 23, 858-867, the entire contents and disclosures of which are hereby incorporated by reference.
Many modes of detection have been employed to monitor separations on microanalytical devices but the most used detection method is laser induced fluorescence (LIF), see Ferrance, J., Landers, J. P., Luminescence 2001, 16, 79-88; and Ferrance, J., Snow, K., Landers, J. P., Clin. Chem. 2002, 48, 380-383, the entire contents and disclosures of which are hereby incorporated by reference. However, the cost of optical instrumentation, the need for analyte derivatization, and the limited portability of LIF has led to the investigation of electrochemical detection (ECD), an attractive alternative for microchip devices, see Verpoorte, E., Electrophoresis 2002, 23, 677-712; Wang, J., Talanta 2002, 56, 223-231; Rossier, J., Reymond, F., Michel, P. E., Electrophoresis 2002, 23, 858-867; Lacher, N. A., Garrison, K. E., Martin, R. S., Lunte, S. M., Electrophoresis 2001, 22, 2526-2536; Vandaveer, W. R. I. V., Pasas, S. A., Martin, R. S., Lunte, S. M., Electrophoresis 2002, 23, 3667-3677; and Wang, J., Trends in Anal. Chem. 2002, 21, 226-232, the entire contents and disclosures of which are hereby incorporated by reference. Since many compounds are electrochemically active, many applications may be found, particularly using direct current (DC) amperometry, see Vandaveer, W. R. I. V., Pasas, S. A., Martin, R. S., Lunte, S. M., Electrophoresis 2002, 23, 3667-3677, the entire contents and disclosure of which is hereby incorporated by reference. However, when a constant potential is applied, the electrode may be fouled by the accumulation of adsorbed carbonaceus material, resulting in an unstable signal, see Fanguy, J. C., Henry, C. S., Analyst 2002, 127, 1021-1023; Garcia, G., Garcia, C. D., Ortiz, P. I., De Pauli, C. P., J. Electroanal. Chem. 2002, 519, 53-59, the entire contents and disclosures of which are hereby incorporated by reference. This effect is particularly detrimental when carbohydrates, thiols or phenols are detected, see Hughes, S., Johnson, D. C., Anal. Chim. Acta 1981, 132, 11-22; and Garcia, C. D., Ortiz, P. I., Electroanalysis 2000, 12, 1074-1076, the entire contents and disclosures of which are hereby incorporated by reference. This problem may be solved with conventional systems by polishing the electrodes to remove build-up or coating the electrodes to prevent build-up, see Kauffmann, J. M., Pekli-Novak, M., Nagy, A., Acta Pharm. Hung. 1996, 66, 57-64; Garcia, C. D., Ortiz, P. I., Electroanalysis 1998, 10, 832-835, the entire contents and disclosures of which are hereby incorporated by reference. To overcome problems associated with electrode fouling in conventional liquid chromatography, a potential waveform referred to as pulsed amperometric detection (PAD) may be applied. In PAD, a high positive potential is applied in order to clean the electrode surface followed by a negative potential step to reactivate the electrode surface. A third, moderate potential is applied for detection of the target analytes. PAD has proven to be effective for a large number of analytes including carbohydrates, amino acids, sulfurs and alcohols, see LaCourse, W. R., Pulsed Electrochemical Detection in High-Performance Liquid Chromatography; Wiley J. & Sons: New York, 1997, the entire contents and disclosure of which is hereby incorporated by reference. PAD is particularly useful when the analyte lacks a strongly-absorbing chromophore (i.e., ultraviolet/visible (UV/Vis) spectrometry) or where other electrochemical techniques are ineffective due to rapid electrode fouling, see Johnson, D. C., Dobberpuhl, D., Roberts, R., Vandeberg, P., J. Chromatogr. 1993, 640, 79-96, the entire contents and disclosure of which is hereby incorporated by reference. Examples where fouling is important include the electrochemical detection of metallic ions, carbohydrates, amines, thiols and alcohols, see Wen, J., Cassidy, R. M., Baranski, A. S., J. Chromatogr., A 1998, 811, 181-192; Lu, W., Cassidy, R., Anal. Chem. 1993, 65, 2878-2881; Deore, B., Yakabe, H., Shiigi, H., Nagaoka, T., Analyst 2002, 127, 935-939; Owens, G. S., LaCourse, W. R., J. Chromatogr., B 1997, 695, 15-25; and LaCourse, W. R., Johnson, D. C., Rey, M. A., Slingsby, R. W., Anal. Chem. 1991, 63, 134-139, the entire contents and disclosures of which are hereby incorporated by reference. These substances are important in a broad range of biological processes and diseases, see Fernandes, J., Saudubray, J. M., Van Den Berghe, G., Inborn Metabolic Diseases: Diagnosis and Treatment, 3rd edition ed., Springer Verlag, 2000, the entire contents and disclosure of which is hereby incorporated by reference. Carbohydrates are not only a significant source of energy for both plants and animals but they also play a substantial role in biological recognition of proteins, see O'Shea, T. J., Lunte, S. M., LaCourse, W. R., Anal. Chem. 1993, 65, 948-951, the entire contents and disclosure of which is hereby incorporated by reference. In addition to being clinically important, amino acids, thiols and alcohols also play an important function assessing the nutritional quality of foods and beverages and give an indication of possible alteration or transformation occurring during food-processing and storage procedures, see Fernandes, J., Saudubray, J. M., Van Den Berghe, G., Inborn Metabolic Diseases: Diagnosis and Treatment, 3rd edition ed., Springer Verlag, 2000; and Casella, I. G., Contursi, M., Desimoni, E., Analyst 2002, 127, 647-652, the entire contents and disclosures of which are hereby incorporated by reference.